Tissue Organ Bath Principles

Tissue Organ Bath Principals 1 RADNOTI TISSUE ORGAN BATH PRINCIPALS Prior to any animal experimentation or use of animal or human tissue, researchers should discuss their protocols with their local animal care and use committee or institutional review board and biosafety (radiation, chemical, biohazard) committees to determine appropriate guidelines for their experiments. The material presented here is believed accurate but no liability is assumed for the use or misuse of said information. Introduction Isolated tissue and isolated organ preparations have been in use for over one hundred years, providing researchers with convenient biological models that exist without the systemic influences of the intact animal. Additionally, isolated tissue preparations can generally be run in groups of 2, 4, 8 or more, are more readily instrumented and can be more easily subjected to controlled changes in perfusate, oxygen drugs and other factors than intact animals. The recent development of transgenic animal models has extended the scope of isolated tissue and organ preparations by creating models that can express normal or pathological human or non-host animal genetic sequences (1). In turn, isolated preparations can permit molecular biologists to quantitate the physiological impact of the expression of these altered genetic sequences at the tissue and organ level. Basic principles overview To create a useful experimental isolated preparation one must construct a system that permits optimal performance of the tissue in a controlled environment that can be suitably instrumented to record changes in tissue function that are of interest to the experimenter. The researcher should therefore first determine (a) the basic hypotheses and specific aims of the research project (b) the species and the types of tissues that would be suitable research models (c) the kinds of experimental responses that might occur in these models (d) the methods, instruments, and statistics required to measure and analyze these responses. A careful review of the literature combined with a thoughtful analysis of the problem is paramount in designing a successful research project. There are numerous articles and reviews of isolated tissue and organ preparations available; a few overviews are listed. The basic requirements for any isolated tissue preparation include provisions for temperature control and oxygen and substrate delivery. A water jacketed organ bath provides a stable and readily adjustable means of temperature control over the normal temperature range of most multicellular preparations. Substrates and other components required to maintain tissue function are usually provided in an aqueous solution, similar in chemical composition to that of the plasma of the donor animal. Oxygen delivery is usually supplied via saturation of the aqueous solution with a gas mixture, either through direct aeration of the solution or, when proteins, blood cells or other readily denatured membrane oxygenator or other non-turbulent delivery system. Additional tissue requirements may include electrical stimulation, changes in flow, pressure or distention, the presence of other cell types or tissues or other specialized features. Instrumentation varies with the type of tissue and phenomena to be observed. For example, instruments are available to detect changes in force, flow or pressure, the release of chemical substances into the perfusate or alteration of perfusate characteristics, oxygen consumption or carbon dioxide production, changes in endogenous or exogenous optical sensors, electrophysiological alterations or literally any type of response the preparation may provide or the investigator may initiate. The responses can be recorded manually or on chart recorders, computers 2 or other devices. Selection of physiological buffer solution and manner of aeration The buffer solution(s) chosen for the dissection and maintenance of the tissue will have a profound effect on the viability of the preparation. Whole blood from donors may be used, but since whole blood is generally in limited supply, it must be recycled, for example with an appropriate peristaltic pump through a membrane oxygenator, without denaturing blood proteins or causing erythrocyte damage. Whole blood may also contain biologically active materials, such as hormones and various cell types, that may affect the tissue preparation. Because of these limitations, most isolated tissue preparations use an artificial plasma-like buffer solution to maintain cell viability. Since almost all of these solutions are water based, water purity is a primary concern. The water purity should at least be the equivalent of double distilled water with organic contaminants removed. Achieving this standard used to be difficult, but with the advent of modern reverse osmosis systems or glass stills passing on water to charcoal and ion exchange resin filters, sources of purified water are now readily available with resistivity measurements in excess of 10MW. Some recipes for buffer solutions still add 0.1-5 mM EDTA (see Table 1) for chelation of heavy metals that may be added from salts or leached from metal tubing or other sources. After storing the water in clean, non leaching containers, the next decision to be made is which formulation of the buffer solution should be used. Reagents should be of high quality (the equivalent of USP or Analytical Reagent grade) and kept isolated from the rest of the chemicals in the laboratory to reduce the possibility of misuse or contamination. There are a number of widely used physiological salt solutions, such as Ringer’s (5), Tyrode’s (6) and Krebs-Hanseleit (7, modified as in 8, see Table 1) and their salt concentrations are often modified to resemble those in the donor’s plasma (see Radnoti Isolated Perfused Heart Table 1 ). Since these solutions must be buffered, a choice of buffering agents must also be made. Bicarbonate buffers such as Tyrode’s and Krebs-Hanseleit, which are based on those naturally occurring in mammalian blood, are effective when in equilibrium with relatively high levels of carbon dioxide, hence the use of 95% oxygen and 5% carbon dioxide gas mixtures to aerate them. The use of 95-100% oxygen rather than atmospheric levels of -20% is to increase the oxygen content of the solution to compensate for the lack of hemoglobin or other oxygen carriers. In most of these solutions, phosphates (or sulfates) are also added. Besides adjusting the pH and increasing buffering capacity, the presence of carbonates, bicarbonates and phosphates aids in maintaining normal anion homeostasis. The restrictions imposed by bicarbonate buffers are the requirement for a mixed gas aeration and the adverse effects on solubility products created by these anions. As an alternative, buffering capacity can be created through the use of synthetic buffers like HEPES or MES in place of bi-carbonates with 100% oxygen used for aeration. It is also possible to use these synthetic buffers in addition to bicarbonate, and then adjust the sodium balance appropriately to maintain osmolarity. The most common metabolic substrate is glucose, with pyruvate, lactate, and fatty acids also added. To aid in glucose metabolism, insulin may be added. Often salts other than calcium are kept as a concentrated stock in one container and calcium and glucose kept as a concentrated stock in another container and then diluted and combined just prior to use. This is done to reduce precipitation of calcium phosphate and retard bacterial growth by having hyperosmotic solutions. To maintain an appropriate oncotic pressure that will reduce edema, albumin, polyvinylpyrrolidine, dextran or other plasma expanders can be added. Note that the presence of albumin or other readily denatured or poorly soluble compounds will necessitate indirect aeration, such as that provided by a membrane oxygenator, to prevent foaming and precipitation. Always adjust the pH of the solution while it is at the bath temperature selected and when it is normally aerated. 3 Gas flow to the buffer is normally controlled with a two-stage regulator designed for the gas mixture utilized. Flow rates of 0.5-2 liters per minute and/or pressures of 1-2 psi are adequate for most preparations. The aeration stream can be controlled with a needle valve and should be a steady line of fine bubbles that do not cause a pronounced “boiling” effect in the bath. Higher pressures will only serve to increase evaporation, jostle the preparation and create noise in the force trace and may perforate glass frits used for aeration. For thin tissues with a large surface area and low metabolic activity (such as blood vessel strips or rings), aeration using room air may be sufficient; in these cases simple aquarium pumps are a cost effective replacement for compressed air tanks. Selection of tissue donors Sources of tissue may be from living or dead donors, depending upon the availability of the tissue type and the requirements of the experimenter. For tissues obtained from deceased donors, the type of tissue, the manner of death or euthanasia, the time elapsed between death and harvesting the tissue and the temperature that the donor’s body is maintained at will affect the viability of the preparation. In the case of living donors, the selection and composition of anesthesia or euthanasia solutions should be made carefully to reduce the impact upon retrieved tissues. The presence of high concentrations of barbiturates, general anesthetics, ethanol, potassium or other substances used in

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